Project Summary/Abstract Unlike HIV-1 in humans and SIVmac in rhesus macaques (RM), primate immunodeficiency viruses do not cause disease in their natural hosts. As a result, a high priority in HIV/AIDS research is understanding natural host infection. A principal model for studies of natural host infection is Sooty Mangabey (SM) infection by SIVsm (which gave rise to SIVmac in macaques and HIV-2 in humans). Emerging evidence suggests that CD4+ subset targeting may be critical in regulating the outcome of infection, both by restricting infection of critical cell types and targeting replication to more dispensable cells. Thus, it is imperative that mechanisms of CD4+ cell subset tropism and targeting in vivo be understood. Current dogma states that SIVsmm/SIVmac replication in vivo is mediated exclusively by CD4 and the entry coreceptor CCR5. CCR5 expression on CD4+ T cells of SM (and other natural hosts) is exceedingly low, which has been thought to be the principal determinant of tropism and a mechanism restricting target cell infection in vivo. In our preliminary studies we made the novel discovery that CCR5 is dispensable for infection and sustained viremia in SM, and that alternative entry pathways, in addition to CCR5, play a major role in SIVsm natural host infection. This finding indicates that the established paradigm of cell targeting and tropism in this important natural host model requires revision. The main hypothesis of this project is that alternative pathways in addition to CCR5 determine target cell infection in SM in vivo, that alternative coreceptor-expressing cells may provide a more expendable target population for replication in the face of highly restricted CCR5 levels, and that to understand CD4+ T cell subset targeting, including entry and post-entry regulation of infection, it is necessary to define the identity, distribution, regulation and use of SIVsm coreceptor entry pathways on CD4+ cells. The goals of this project are to (1) Identify the alternative coreceptors used by SIV in primary SM CD4+ cells;(2) Determine how alternative coreceptor expression, in conjunction with CCR5, is regulated and distributed on specific subsets of blood, lymphoid &mucosal CD4+ T cells (Tcm, Tem, Th17, Teff, etc);(3) Define the role of alternate coreceptor and CCR5 in determining SIVsm/SIVmac target cell infection in vivo, and;(4) Identify postentry restrictions that may regulate infection of alternative coreceptor/CCR5-expressing CD4+ T cell subsets. This work will significantly alter current concepts of natural host infection and addresses a novel hypothesis for mechanisms underlying nonprogressive infection in these animals, and will provide essential information necessary in order to effectively use this important model to better understand pathogenesis.